Presence of B Cells and Plasma Cells in Oral Lichen Planus

Statement of the Problem: Oral lichen planus (OLP) is a chronic inflammatory disease with unknown etiopathogenesis. It was believed that T cells played the major role in developing the lesions. It has been recently suggested that B lymphocyte cells (B cells) and plasma cells may play a role in OLP pathogenesis. Purpose: OLP is considered as a T-cell mediated disease. It was believed that the presence of B cells and plasma cells in the sub-epithelial inflammatory infiltrate, rules out the diagnosis of OLP. This study aims to investigate the presence of B cells and plasma cells in the inflammatory infiltrate of OLP. In addition, the association between the presence of B cells and plasma cells with histopathologic features of the lesion was assessed. Materials and Method: To assess the presence of B cells and plasma cells, 61 cases with the diagnosis of OLP were collected. The cases with definite clinical and histopathological diagnosis of lichen planus based on WHO criteria were included. For each case, demographic information and histological characteristics were recorded. Specimens underwent immunohistochemical (IHC) staining for CD20 and CD138 and the percentage of the positive cells were counted and scored. Results: CD20 positive cells existed in all OLP cases with the mean expression of 22.5%± 15.17% and small number of CD138 positive cells were seen in 62.3% of our cases with the mean expression of 4.74%±9.23%. No association was found between histopathological features and CD138 expression, however, CD20 expression level was higher in the cases with parakeratinized surface (p= 0.004). Conclusion: B cells existed in the inflammatory infiltrate of OLP in all cases. Small number of plasma cells could be occasionally found in OLP. Therefore, presence of B cells and plasma cells in the inflammatory infiltrate cannot rule out the diagnosis of OLP.

immune reaction to keratinocytes. The characteristics of band-like lymphocytic infiltration are found to be a clue to etiopathogenesis of OLP [6,[8][9].
Oral mucosa is exposed to variety of allergic materials such as dental restorative and casting materials like dental amalgam and Nickel. Foods like cinnamon and drugs are known to induce allergic reaction in the oral mucosa namely oral lichenoid reaction (OLR). Although OLR lesions are indistinguishable from OLP both clinically and histopathologically, unlike OLP, they do not go under malignant transformation. Moreover, contrasting OLP, lichenoid reactions secondary to dental restorative materials, medications, or foods can be resolved by changing restorative material or drug or food habits. Briefly, OLP and OLR are two distinct lesions with different causes that need different considerations [10][11]. Many studies have investigated the content of inflammatory infiltrate to distinguish OLR from OLP. Some researchers have attributed the presence of B-lymphocytes (B cell) and plasma cells in the lymphocytic infiltrate as one of the definitions of OLR lesions [12][13][14]. CD20 is a phosphor-protein expressed on B cells from pre B stage to the late stage of maturation.
The expression decreases as B cells turn into plasma cells [15].
CD138 (syndecan-1) is a proteoglycan that facilitates cell-to-cell adhesion, cell and extra-cellular matrix interaction, cell differentiation and proliferation. CD138 is present on mature epithelial cells and plasma cells while is absent on endothelial and normal mesenchymal cells. Based on messenger RNA studies, CD138 is highly expressed on normal and neoplastic plasma cells and is absent on other cell types [16][17][18].
The aim of this study was to assess the presence of B cells and plasma cells in the inflammatory infiltrate of OLP by immunohistochemical (IHC) staining of CD20 and CD138 respectively. Moreover, the association between the presence of B cells and plasma cells with histopathologic features of the lesion was evaluated.

Materials and Method
This study cross-sectional study was conducted in Oral

Evaluation of Hematoxylin & Eosin (H&E) slides
The sections were reviewed by an oral and maxillofacial pathologist to determine the histopathologic features as follows: keratosis, acanthosis, granulosis, spongiosis, hydropic degeneration of basal layer, lymphocyte exocytosis, epithelium separation, intensity of inflammation (mild, moderate, and severe) and the degree of epithelial dysplasia (no, mild, moderate, severe and SCC) [20]. They were left for 1 hour in room temperature, and then treated with PBS. Diaminobenzidine solution (Vector, Burlingame, CA, USA) 0.3% was used to visualize reaction products. As the last step, the sections were coun-terstained with Mayer's hematoxylin, dehydrated, and mounted. Normal tonsillar tissue was included as positive control for CD20. For CD138 internal control was used [9]. For negative control, sections were treated with normal saline and were confirmed to be unstained. Tonsillar tissue served as external control for CD20 and membranous staining of the oral epithelial cells served as the internal control for CD138.

Scoring
For both markers, each section was examined by light microscope (OLYMPUS, BX51) at the magnitude of 200× in 10 randomly selected fields. The number of cells in the band-like inflammatory infiltration with membranous staining were counted and the percentage of stained cells were reported as grade 1 for no expression, grade 2 for less than 50% expression, and grade 3 for more than 50% expression [9].

Results
Of the 61 specimens, 38(62.3%) belonged to female cases and 23(37.7%) belonged to male cases. The mean age of the patients was 49.6 years old. Buccal mucosa was the most frequent site of involvement (83.6%), and palate was the least frequent site with the frequency of 1.6 ( Table 1). Epithelial dysplasia was found in 15 cases (24.6%). CD20 immuno-expression was found in all cases ( Figure 1) while CD138 was expressed in 38 (62.3%) of cases ( Figure 2). The mean expression of CD20 and CD138 were found to be 22.5%±15.17% and 4.74%±9.23%, respectively (Table 2).  No association was found between histopathological features and CD138 expression, however, CD20 expression level was higher in the cases with parakeratinized surface (p=0.004). The correlation between CD20 and CD138 was statistically significant (coefficient= 0.317 p= 0.013).

Discussion
OLP is a chronic oral mucosal disease with autoimmune entity due to T cell lymphocytes predominance. The exact etiology and pathogenesis is unknown [21]. Many   indicative of OLR [10][11][12]. In this research, we con-  [22]. Mravak-Stipetić et al. [23] showed that plasma cells were present in both OLP This finding is a newly discovered factor to OLP pathogenesis. We used CD20 to assess the presence of B cells in OLPs. CD20 is present on all maturation stages of B cell from pre B cell to immature and activated B cell [15]. There are some CD markers such as CD27 and CD5, which appear on special stages of maturation, and their assessment would be helpful in determining the type of B cell in lymphocytic infiltrate [9].
We suggest other future studies to evaluate the presence of EBV and other viral infections in sections to check their association with plasma cells.